The present invention relates to magnetic resonance (MR) analysis, particularly to magnetic resonance spectroscopy (MRS) and imaging (MRI) of biological objects.
Magnetic resonance is a phenomenon exhibited by a select group of atomic nuclei and is based upon the existence of nuclear magnetic moments in these nuclei (termed xe2x80x9cgyromagneticxe2x80x9d nuclei). When a gyromagnetic nucleus is placed in a strong, uniform and steady magnetic field (a so-called xe2x80x9cexternal fieldxe2x80x9d and referred to herein as a xe2x80x9cstaticxe2x80x9d magnetic field), it precesses at a natural resonance frequency known as a Larmor frequency. The Larmor frequency is characteristic of each nuclear type and is dependent on the applied field strength in the location of the nucleus. Typical gyromagnetic nuclei include 1H (protons), 13C, 19F and 31P. The resonant frequencies of the nuclei can be observed by monitoring the transverse magnetization that results after a strong RF pulse applied at or near their Larmor frequencies. It is common practice to convert the measured signal to a frequency spectrum by means of Fourier transformation.
More specifically, when a bulk sample containing nuclear magnetic resonance (NMR) active nuclei is placed within a magnetic field, the nuclear spins distribute themselves amongst the nuclear magnetic energy levels in accordance with Boltzmann""s statistics. This results in a population imbalance between the energy levels and a net nuclear magnetization. It is this net nuclear magnetization that is studied by NMR techniques.
At equilibrium, the net nuclear magnetization is aligned parallel to the external magnetic field and is static. A second magnetic field perpendicular to the first and rotating at, or near, the Larmor frequency can be applied to induce a coherent motion of the net nuclear magnetization. Since, at conventional field strengths, the Larmor frequency is in the megahertz frequency range, this second field is called a xe2x80x9cradio frequencyxe2x80x9d or RF field.
In particular, a short (microsecond) pulse of RF radiation is applied to the sample in the static magnetic field; this pulse is equivalent to a range of frequencies. The free induction decay (FID) in response to the RF pulse is measured as a function of time. The response of the sample to the pulse depends upon the RF energy absorption of the sample over a range of frequencies applied (for example, 500 MHzxc2x12500 Hz). Often the pulse is applied many times and the results averaged to improve the signal-to-noise ratio.
The coherent motion of the nuclear magnetization about the RF field is called a xe2x80x9cnutation.xe2x80x9d In order to deal conveniently with this nutation, a reference frame is used which rotates about the z-axis at the Larmor frequency. In this xe2x80x9crotating framexe2x80x9d the RF field, which is rotating in the stationary xe2x80x9claboratoryxe2x80x9d reference frame, is static. Consequently, the effect of the RF field is to rotate the nuclear magnetization direction at an angle with respect to the main static field direction. By convention, an RF field pulse of sufficient length to nutate the nuclear magnetization through an angle of 90xc2x0 or xcfx80/2 radians is called a xe2x80x9cxcfx80/2 pulse.xe2x80x9d
A xcfx80/2 pulse applied with a frequency near the nuclear resonance frequency will rotate the spin magnetization from an original direction along the main static magnetic field direction into a plane perpendicular to the main magnetic field direction. Because the RF field and the nuclear magnetization are rotating, the component of the net magnetization that is transverse to the main magnetic field precesses about the main magnetic field at the Larmor frequency. This precession can be detected with a receiver coil that is resonant at the precession frequency and located such that the precessing magnetization induces a voltage across the coil. Frequently, the xe2x80x9ctransmitter coilxe2x80x9d employed for applying the RF field to the sample and the xe2x80x9creceiver coilxe2x80x9d employed for detecting the magnetization are one and the same coil.
In addition to precessing at the Larmor frequency, in the absence of the applied RF field, the nuclear magnetization also undergoes two relaxation processes: (1) the precessions of various individual nuclear spins which generate the net nuclear magnetization become dephased with respect to each other so that the magnetization within the transverse plane loses phase coherence (so-called xe2x80x9cspin-spin relaxationxe2x80x9d) with an associated relaxation time, T2, and (2) the individual nuclear spins return to their equilibrium population of the nuclear magnetic energy levels (so-called xe2x80x9cspin-lattice relaxationxe2x80x9d) with an associated relaxation time, T1. The spin-spin relaxation is caused by the presence of small local magnetic fields, arising from the electrons and magnetic nuclei surrounding a particular nucleus. These fields cause slight variations in the resonance frequency of the nuclei, which results in a broadening of the NMR resonance line. Often this broadening is caused by two types of local fields: a static component, which gives rise to a so-called inhomogeneous broadening, and local fields which are fluctuating in time as a result of molecular motions. The latter phenomenon results in a so-called homogeneous broadening.
Magnetic resonance imaging and magnetic resonance spectroscopy are used extensively in biological research and medicine, both for in vitro investigations of cells and tissues and for in vivo measurements on animals and humans. Both methods are used for a large variety of applications, including the detection and diagnosis of lesions and diseases, and the evaluation of therapy response. One particularly useful MRS technique is 1H nuclear magnetic resonance (NMR) spectroscopy. 1H NMR spectroscopy has been used extensively to study metabolic changes in diseased cells and tissues and the effects of therapy. The resonance lines corresponding to several key mobile compounds have been observed, and their spectral intensities have been linked to the tumor phenotype, tumorigenesis, tumor size, increased proliferation of cells, cell apoptosis, and necrosis.
However, a serious problem associated with these applications is the relatively large widths of the MR resonance lines that are observed using conventional MRI and MRS. This reduces the MRI and MRS sensitivity, and, for MRS, can result in severely overlapping spectral lines, which seriously hampers the analysis of the spectrum. It has been established that in biological materials the line widths are mainly caused by inhomogeneous broadening. In intact cells and tissues, the possible mechanisms that broaden the lines inhomogeneously include residual chemical shift anisotropy interaction and local magnetic field gradients arising from variations in the bulk magnetic susceptibility at the various compartment boundaries present in the cells and tissues. It is believed in the art that the bulk magnetic susceptibility variations are the main mechanisms responsible for the broadening. Using cell extracts can eliminate this broadening, but this procedure is time consuming and may introduce spectral artifacts.
It is well known that the susceptibility broadening and other inhomogeneous broadening mechanisms can be eliminated by magic angle spinning (MAS), where the sample is rotated about an axis with an angle of 54xc2x044xe2x80x2 (or cosxe2x88x921 (3xe2x88x921/2)) with respect to the static magnetic field direction. A problem with MAS is that when the value of the spinning rate is small compared to the width of the broadening, the resonant peak splits into a group of spinning sidebands (SSBs) separated by the spinning rate. If the value of the spinning rate is less than the isotropic spectral width, the analysis of the spectra becomes considerably difficult due to the overlapping of the SSBs associated with the different resonant peaks. This problem can be avoided by increasing the spinning rate to eliminate the SSBs in the spectral region of interest. Indeed it has been shown that fast MAS, where a sample is rotated at a speed of several kHz, produces a significant narrowing of the MR lines in cells and tissues (see Weybright et al., Gradient, High-Resolution, Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy of Intact Cells, Magnetic Resonance in Medicine 1998; 39: 337-345; and Cheng et al., Quantitative Neuropathology by High Resolution Magic Angle Spinning Proton Magnetic Resonance Spectroscopy, Proc. Natl. Acad. Sci. USA 1997; 94: 6408-6413). However, the large centrifugal force associated with such high spinning rates destroys the tissue structure and even part of the cells (see Weybright et al.). Consequently, MAS at a high spinning speed is not suitable, for example, to map the metabolite distribution in intact biological tissues or to study live cells, and is impossible to use on live subjects.
A possible way to overcome the problems associated with fast MAS is to use slow sample spinning. Many methods have been developed in solid state NMR to eliminate the spinning sidebands or to separate them from the isotropic spectrum so that a sideband free isotropic chemical shift spectrum is obtained. One approach is the so-called magic angle turning (MAT) techniques, and sideband free isotropic chemical shifts have been obtained in solids at spinning rates as low as 30 Hz (Hu et al., Magic Angle Turning and Hopping, in Encyclopedia of Magnetic Resonance D. M. Grant, and R. K. Harris, Eds. New York: John Wiley and Sons: 1996, 2914-2921).
MAT is a two dimensional (2D) NMR technique that was developed to determine the chemical shift tensors of rare spins such as 13C and 15N in solids. There are basically two types of MAT experiments. The first type (MAT-1) is based on the Magic Angle Hopping (MAH) experiment pioneered by Bax et al., Correlation of Isotropic Shifts and Chemical Shift Anisotropies by Two-Dimensional Fourier-Transform Magic-Angle Hopping NMR Spectroscopy, J. Magn. Reson. 1983; 52: 147. The second class (MAT-2) involves the use of five radio frequency xcfx80 pulses during a constant evolution time period (e.g., one rotor period). MAT-2 techniques include the five xcfx80 replicated magic angle turning (FIREMAT) (Hu et al., An Isotropic Chemical Shift-Chemical Shift Anisotropy Magic Angle Slow-Spinning 2D NMR Experiment, J. Magn. Reson. 1993; A 105: 82-87; and Alderman et al., A Sensitive, High Resolution Magic Angle Turning Experiment for Measuring Chemical Shift Tensor Principal Values, Molecular Physics 1998; 95(6): 1113-1126) and the 2D-phase-altered spinning sidebands (PASS) techniques (Antzutkin et al., Two-Dimensional Sideband Separation in Magic-Angle-Spinning NMR,. J. Magn. Reson 1995; A115: 7-19). All of these experiments are 2D isotropic-anisotropic chemical shift correlation experiments yielding a high resolution isotropic chemical shift dimension and a chemical shift anisotropy dimension. Although MAT has been applied in solid state NMR (see Hu et al., Magic Angle Turning and Hopping; Gan et al., High-Resolution Chemical Shift and Chemical Shift Anisotropy Correlation in Solids Using Slow Magic Angle Spinning, J. Am. Chem. Soc. 1992; 114: 8307-8309; Hu et al., Magic-Angle-Turning Experiments for Measuring Chemical-Shift-Tensor Principal Values in Powdered Solids, J. Magn. Reson. 1995: A 113: 210-222; Hu et al., An Isotropic Chemical Shift-Chemical Shift Anisotropy Magic Angle Slow-Spinning 2D NMR Experiment; Alderman et al., A Sensitive, High Resolution Magic Angle Turning Experiment for Measuring Chemical Shift Tensor Principal Values; and Antzutkin et al., Two-Dimensional Sideband Separation in Magic-Angle-Spinning NMR), its potential for biological research has not been explored.
One of the reasons that MAT for biological objects, as opposed to solid objects, has not been investigated is the belief that the diffusion of the molecules containing the nuclei of interest in the internal static local magnetic fields results in a time-dependent field as experienced by the nuclei. This effect worsens if the spinning frequency is reduced, resulting in imperfect suppression of the SSB""s. Indeed, it was shown that a sideband-suppression technique called total suppression of sidebands (TOSS) was ineffective for suppressing SSB""s arising from water embedded in glass beads when the spinning speed was lowered (see Liu et al, Manipulation of Phase and Amplitude Modulation of Spin magnetization in Magic Angle Spinning NMR in the Presence of Molecular Diffusion, J. Chem. Phys. 2001; 114: 5729-5734).
Another approach for increasing the sensitivity and resolution of NMR spectroscopy involves rotating the magnetic field around the sample rather than the sample around the magnetic field. According to this approach the sample remains stationary. For example, Bradbury et al., Nuclear Magnetic Resonance in a Rotating Magnetic Field, Phys. Letters 1968; 26A: 405-406, disclose rotating the static magnetic field by superposing a static field and two sinusoidal fields in phase quadrature in the plane perpendicular to the static field and with amplitudes that are a factor 2 larger than that of the static component. However, this approach was never considered any further.
Thus, a need exists for a method for obtaining high resolution magnetic resonance analysis of biological objects. In particular, there is a need for a magnetic resonance analysis technique that does not damage tissue or cell structure in biological objects and avoids the problems associated with SSBs at slow object spinning rates.
The present invention provides a method for magnetic resonance analysis of an object by combining slow magic angle techniques with certain radio frequency pulse sequences. This combination provides for the first time a method for obtaining high resolution spectra of a biological object that (a) does not damage tissue or cellular structure in the biological object and (b) substantially eliminates spinning sideband peaks in the spectra associated with slow magic angle spinning. Contrary to the conventional expectation that the diffusion of the molecules containing the nuclei of interest in the internal static local magnetic fields would be problematic for slow spinning, the inventors have surprisingly discovered that the inventive method provides SSB suppression at low rotation frequencies.
In particular, according to a first embodiment of the invention there is provided a method of performing a magnetic resonance analysis of a biological object that includes placing the biological object in a main magnetic field and in a radio frequency field, the main magnetic field having a static field direction; rotating the biological object at a rotational frequency of less than about 100 Hz around an axis positioned at an angle of about 54xc2x044xe2x80x2 relative to the main magnetic static field direction; pulsing the radio frequency to provide a sequence that includes a magic angle turning pulse segment; and collecting data generated by the pulsed radio frequency.
According to a second embodiment of the invention there is provided a method of performing a magnetic resonance analysis of a biological object that includes placing the biological object in a main magnetic field and in a radio frequency field, the main magnetic field having a static field direction; rotating the biological object at a rotational frequency of less than about 100 Hz around an axis positioned at an angle of about 54xc2x044xe2x80x2 relative to the main magnetic static field direction; pulsing the radio frequency to provide a sequence capable of producing a spectrum that is substantially free of spinning sideband peaks; and collecting data generated by the pulsed radio frequency.
According to a third embodiment of the invention there is provided a method of performing a magnetic resonance analysis of a biological object that includes subjecting the biological object to a static magnetic field and a pulsed radio frequency field, the main magnetic field having a static field direction; rotating the biological object at a rotational frequency of less than about 100 Hz around an axis positioned at an angle of about 54xc2x044xe2x80x2 relative to the main magnetic static field direction; controlling the pulsed radio frequency to provide a sequence of pulses of radio frequency radiation capable of producing a spectrum that is substantially free of spinning sideband peaks; and generating a magnetic resonance analysis of the response by nuclei in the biological object to the pulsed radio frequency sequence.
According to a fourth embodiment of the invention there is provided a method of performing a magnetic resonance analysis of a biological object that includes placing the biological object in a main magnetic field and in a radio frequency field, the main magnetic field having a static field direction; positioning the object along a magic axis located at an angle of about 54xc2x044xe2x80x2 relative to the main magnetic static field direction; reorienting the main magnetic field about the magic angle axis between three predetermined positions at a rotational frequency of less than about 100 Hz, the three predetermined positions being related to each other by 120xc2x0; pulsing the radio frequency to provide a sequence capable of producing a spectrum that is substantially free of spinning sideband peaks; and collecting data generated by the pulsed radio frequency.
According to a fifth embodiment of the invention there is provided a method of performing a magnetic resonance analysis of a biological object that includes providing a main magnetic field that includes a first component having a static field direction and an amplitude and a second and a third component, each second and third component having a sinusoidal field in a plane perpendicular to the static field direction of the first component and with an amplitude that is 21/2 times the amplitude of the static field of the first component, wherein the second and third components produce a magnetic field that rotates at a frequency of less than about 100 Hz around an axis located at an angle of about 54xc2x044xe2x80x2 relative to the static field direction of the first component; placing the biological object in the main magnetic field and in a radio frequency field; pulsing the radio frequency to provide a pulse sequence capable of producing a spectrum that is substantially free of spinning sideband peaks; and collecting data generated by the pulsed radio frequency.
The methods of the invention are especially useful for increasing the NMR sensitivity in a MRI experiment, and for increasing the sensitivity and resolution of NMR spectra of 1H and other NMR-sensitive nuclei in MRS experiments in intact excised tissues and organs and in live animals and humans. The slower rotating of the sample according to the present invention minimizes, if not substantially eliminates tissue and cellular damage. The invention has several important advantages over fast MAS: (I) larger rotors and, henceforth, larger samples can be used, which increases the NMR sensitivity (especially important when the method is applied for less NMR-sensitive nuclei than protons); (II) the structural integrity of the biological sample undergoes minimal or no changes under slow spinning (i.e., artifacts in the spectra induced by the fast spinning, which are a result of the sample deformation during the spinning, are avoided); and (III) besides the isotropic spectrum, the anisotropy patterns of the individual water and metabolite lines can be determined (allowing one to obtain information regarding the immediate surroundings of the various compounds).